RNA isolation from soluble urine fractions

ABSTRACT

The invention provides methods for isolating RNA from the soluble fraction of urine. The methods can be used for detecting the presence or absence of an RNA, or quantifying the amount of an RNA. The methods are useful for diagnosing an individual suspected of having a disease by detecting the level of RNA associated with the disease in the soluble fraction of urine. The methods are also useful for prognosing an individual diagnosed with a disease by detecting the level of RNA associated with the disease in the soluble fraction of urine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/130,039, filed Apr. 15, 2016, now issued U.S. Pat. No. 10,066,226,which is a Continuation of U.S. application Ser. No. 12/973,747, filedDec. 20, 2010, now issued U.S. Pat. No. 9,315,802, which claims benefitof U.S. Provisional Application No. 61/290,976, filed Dec. 30, 2009.

FIELD OF THE INVENTION

This invention relates to methods for isolating, processing, andidentifying nucleic acids from biological fluids.

SUMMARY OF THE INVENTION

The present invention relates to methods for extracting RNA from urine.In some embodiments, the a soluble urine concentrate is formed from theurine sample. In other embodiments, the urine sediment is removed fromsoluble urine concentrate. Optionally, the RNA is subsequently processedin order to determine the identity, presence, or amount of the RNA orthe gene products encoded by that RNA, identify the cell type(s) fromwhich the RNA originated, and/or to determine a diagnosis or prognosisfor a pathological conditions indicated by the presence and/or identityof that RNA. Preferably, the RNA extracted from the soluble fraction ofurine is mammalian RNA (e.g., non-viral RNA). Suitable mammalian urinesample include, for example, urine samples obtained from humans, horses,dogs, and cats. Optionally, the subject from which the urine sample isobtained has been previously diagnosed as having a pathologicalcondition.

Generally, the methods provide for isolating RNA from the solublefraction of urine from an individual (e.g., a patient) by concentratingthe RNA in the urine sample to produce a soluble urine concentrate. TheRNA may be subsequently isolated from the soluble urine concentrate ormay be further processed without isolation. Optionally, the urinesediment may be separated from the soluble urine fraction prior toconcentration. Alternatively, the urine sediment may be physically orfunctionally separated from the soluble urine fraction during theconcentration step. Urine sediment that is functionally separated fromthe soluble urine fraction may be in physical contact with the solublefraction but has been processed in such a manner that the contents ofthe sediment do not contribute to the RNA of the soluble fraction. Forexample, cells, organelles, other cellular debris, and insoluble matter(e.g., mineral crystals) may be pelleted from the urine sample bycentrifugation and the sample further processed in order that the cellsand organelles are not disrupted to either become resuspended in thesoluble fraction or lysed to release nucleic acids contained thereininto the soluble fraction. Alternatively, the urine sediment may beseparated from the soluble fraction by filtration.

The RNA in the soluble urine fraction may be concentrated to form asoluble urine concentrate by any suitable means including, for example,ultrafiltration (e.g., using a filter membrane having a cutoff of about100 kDa, 50 kDa, 10 kDa, or 3 kDa), lyophilization, dialysis, or othermeans for dehydration or concentration. In some embodiments, the solubleurine concentrate is formed by ultrafiltration of the soluble urinefraction through a membrane filter having a suitable molecular weightcutoff. Ultrafiltration may be performed using a syringe filter or acentrifugal filtration unit. In some embodiments, the membrane filterhas a net positive or net neutral charge. Suitable membrane materialsinclude, for example, cellulose based materials (e.g. regeneratedcellulose, methylcellulose, cellulose triacetate), polysulfone, andpolyethersulfone. Dialysis may be performed against any suitablecounter-solvent including for, example, polyethylene glycol, and thedialysis membranes are designed to have any appropriate molecular weightcutoff, as described herein. In embodiments which use lyophilization toform the soluble urine concentrate, the urine sediment is physicallyseparated from the soluble urine fraction prior to lyophilization.Optionally, the lyophilized product containing the RNA from the solublefraction is resuspended (solubilized) following lyophilization in avolume of diluent less than the original volume of the urine sample. TheRNA from the soluble urine concentrate may be isolated by any suitablemethod including, for example, solid phase extraction. Optionally, theRNA is subsequently released from the solid phase for furtherprocessing.

Specifically, in one aspect, the invention provides a method forisolating RNA from the soluble fraction of urine by: a) concentrating aurine sample to produce a soluble urine concentrate having RNA, and b)isolating RNA from the soluble urine concentrate.

In another aspect, the invention provides a method for determining thepresence or amount of a target RNA in a urine sample, by isolating RNAfrom the soluble fraction of urine using any of the methods describedherein, and using the isolated RNA in a hybridization-based assay todetermine the presence or amount of the target RNA. Target RNA may bedetected and/or quantified using any appropriate method includinghybridization-based methods such as real-time PCR.

In another aspect, the invention provides a method for amplifyingnucleic acids from a urine sample by a) concentrating the urine sampleto produce a soluble urine concentrate, b) isolating RNA from thesoluble urine concentrate produced in step (a), c) reverse transcribingthe RNA from the soluble urine concentrate produced in step (b) to formcDNA; and d) amplifying the cDNA produced in step (c).

In any of these methods, optionally two or more RNAs or theircorresponding cDNAs are detected and/or quantified.

Also provided are methods for determining a diagnosis or prognosis foran individual. The methods producing a soluble urine concentrateaccording to any of the methods provided herein and determining thepresence or identity of the RNA, if any, in that soluble urineconcentrate. For diagnosis, the amount of RNA (either total or of aspecific type) is compared to a reference value (e.g., the amount ofthat RNA present in a healthy individual or an individual known to havethe disease under investigation). For prognosis, the amount or type ofthe RNA may be compared to a reference value, wherein the referencevalue is referenced to either healthy individuals or individuals knownto have specific disease outcomes. Exemplary diseases amenable toanalysis by the methods described herein include, for example, prostatecancer, bladder cancer, uterine cancer, ovarian cancer, and cervicalcancer. In one example, benign prostate hyperplasia may be diagnosed byassessing one or more urinary RNAs selected from the group consisting ofheat shock 60 kDa protein (HSPD1), inosine monophosphate dehybrogenase 2(IMPDH2), PDZ and LIM domain 5 (PDLIM5), and UDP-N-acteylglucosaminepyrophosphorylase 1 (UAP1).

In one example, the above methods further include detecting the RNA fromthe soluble urine concentrate. In one example of the above methods, theRNA is mRNA. Exemplary methods for detecting RNA include reversetranscription coupled with real-time PCR, northern blot, UVspectroscopy, hybridization of RNA or cDNA to a probe such as inmicroarray or flow cytometry.

Urine may be obtained from any individual. An individual may be healthyand without any known disease. Alternatively, an individual may be aperson suspected of having a disease. Urine samples may be pooled frommultiple individuals or from multiple samples obtained from a singleindividual. In the latter case, the combined sample may represent thetotal daily urinary output of a single individual. Preferably, urinesamples are collected in sterile containers in order to minimize thepossibility for contamination by environmental microorganisms or otherforeign matter. In one embodiment, the urine sample is obtained using acatheter.

As used herein the term “soluble fraction of urine” means urine which issubstantially free (less than about 1% w/w) of cells, cellular debris,organelles, organisms, and insoluble matter (e.g., mineral crystals).Typically, an unprocessed urine sample obtained from an individual is amixture of the soluble fraction and the urine sediment, with the solublefraction making up the largest portion of the mixture. Under normalconditions, the material that makes up the urine sediment is suspendedin the soluble fraction and requires processing to effect usefulseparation. Preferably, the soluble fraction of urine and the resultingsoluble urine concentrate are acellular (i.e., lacking cells). It isunderstood that the urine fraction may be rendered acellular (e.g., byfiltration and/or centrifugation) without necessarily removing all otherinsoluble matter such as organelles, cellular debris, and insolublematter.

As used herein the term “urine sediment” means that fraction of urinecomprising cells, cellular debris, organelles, organisms, and/orinsoluble matter that may be removed from the soluble fraction of urine.Exemplary methods for separating urine sediment from soluble fraction ofurine include centrifugation, filtration, and/or sedimentation undergravity.

As used herein the term “soluble urine concentrate” means that solublefraction of urine which is substantially free (less than about 1% w/w)of cells, cellular debris, organelles, and organisms and where thevolume of the soluble fraction of urine has been reduced by at least50%, at least 60%, at least 75%, at least 80%, at least 90% or more fromthe original urine volume.

As used herein the term “ultrafiltration” means a separation processwhich includes a filtration through a semi permeable membrane under apositive pressure such that solutes of higher molecular weight areretained by the membrane while water and low molecular weight solutespass though the membrane. Exemplary positive pressure includes but notlimited to hydrostatic pressure, centrifugal force.

As used herein the term “nominal molecular weight limit” in the contextof a filter membrane means a pore size where over 90% of the solute withthat molecular weight will be retained. Exemplary nominal molecularweight limit suitable for concentrating soluble urine fractioncomprising RNA include 3 kDa, 10 kDa, 30 kDa, 50 kDa and 100 kDa.

The term “RNA” is meant to include mRNA, tRNA, and rRNA. In preferredembodiments, the RNA is mammalian RNA (e.g., RNA obtained from mammalianurine). In other embodiments, the RNA is non-viral.

“Primer” refers to an oligonucleotide that hybridizes to a substantiallycomplementary target sequence and is capable of acting as a point ofinitiation of DNA synthesis when placed under conditions in which primerextension is initiated (e.g., primer extension associated with anapplication such as PCR). An oligonucleotide “primer” may occurnaturally, as in a purified restriction digest or may be producedsynthetically. Primers are typically between about 10 and about 100nucleotides in length, preferably between about 15 and about 60nucleotides in length, more preferably between about 20 and about 50nucleotides in length, and most preferably between about 25 and about 40nucleotides in length. An optimal length for a particular primerapplication may be readily determined in the manner described in H.Erlich, PCR Technology, Principles and Application for DNA Amplification(1989).

A “probe” refers to an oligonucleotide that interacts with a targetnucleic acid via hybridization. A probe may be fully complementary to atarget nucleic acid sequence or partially complementary. The level ofcomplementarity will depend on many factors based, in general, on thefunction of the probe. A probe or probes can be used, for example todetect the presence or absence of an RNA or cDNA in a sample by virtueof the sequence characteristics of the target. Probes can be labeled orunlabeled, or modified in any of a number of ways well known in the art.A probe may specifically hybridize to a target nucleic acid. Probes canbe designed which are between about 10 and about 100 nucleotides inlength and hybridize to the target nucleic acid such as RNA or cDNA.Oligonucleotides probes are preferably 12 to 70 nucleotides; morepreferably 15-60 nucleotides in length; and most preferably 15-25nucleotides in length. The probe may be labeled with a detectable label.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the experimental design of isolating RNAfrom urine sediment, urine filtrate (after removing cells from urine byfiltration) and whole urine. Three different RNA isolation proceduresare illustrated.

FIG. 2 is a bar graph showing the relative recovery of RNA from wholeurine and the supernatant and sediment fractions, using RT-PCR,following different time-to-process delays after urine collection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for isolating RNA from thesoluble fraction of urine. Also provided are methods for detecting RNAin soluble fraction of urine and methods for diagnosis and prognosis bydetecting RNA associated with a disease in soluble fraction of urine.

Sample

A urine sample typically consists of a soluble fraction and a sedimentfraction. The sediment fraction may contain cells, cellular debris,organelles, microorganisms, and/or insoluble minerals (e.g., kidneystones). Soluble urine fraction is substantially free (less than 1% w/w)of urine sediment and preferably contains only soluble molecules (e.g.,urea, nucleic acids, soluble proteins, etc.). Urine samples may beobtained from healthy individuals (i.e., free of known disease) orindividuals known or suspected to have a disease or other condition.Alternatively, a urine sample may consist of urine samples pooled fromseveral individuals.

Methods for Separating Urine Sediment From Soluble Urine Fraction

The urine sediment may be separated from the soluble urine fraction byany convenient method including, for example, centrifugation,sedimentation under gravity, or filtration. In one example,centrifugation can be performed at 1000× g to 30,000× g for 10 minutesto pellet the urine sediment and some or all of the soluble urinefraction may be removed. In another example, urine sediments can beseparated by filtration using relatively high molecular weight cutofffilters such that the urine sediment is retained on the filter membranewhile the soluble urine fraction including the soluble RNA passes intothe filtrate. Exemplary filter membranes can be made of cellulose basedmembranes (e.g. regenerated cellulose, methylcellulose, cellulosetriacetate), polysulfone, polyethersulfone. Commercial kits such as ZRUrine RNA Isolation Kit™ (ZYMO Research Corporation) are available toremove urine sediments from soluble urine fraction.

Methods for Concentrating Soluble Urine Fractions

Soluble urine fractions may be concentrated by any convenient methodsuitable for the volume of urine to be processed and the anticipatedsize of the soluble RNA to be identified and isolated. Suitableconcentration methods include, for example, ultrafiltration,lyophilization, and dialysis (e.g., against polyethylene glycol).Ultrafiltration, involves filtration though a semi permeable membraneunder a positive pressure such as hydrostatic pressure or centrifugalforce such that solutes of higher molecular weight remain in theretentate while water and low molecular weight solutes pass into thefiltrate. Typically, the membranes used for concentration have a smallerpore diameter (e.g., lower molecular weight cutoff) than the filtersused to remove the urine sediment.

Preferably, the semipermeable membrane materials used for concentratingsoluble urine fractions do not bind or retain soluble RNA. Suitablematerials include, for example, cellulose based materials (e.g.regenerated cellulose, methylcellulose, cellulose triacetate),polysulfone, and polyethersulfone. The semipermeable membranes areavailable in various pore sizes. The pore size where over 90% of thesolute with that molecular weight will be retained is termed as “nominalmolecular weight limit” (NMWL). Exemplary nominal molecular weightlimits suitable for isolating RNA from urine include 3 kDa, 10 kDa, 30kDa, 50 kDa and 100 kDa. The pore size of semipermeable membranesinclude nominal molecular weight limits that can range from about 1 kDato about 200 kDa, from about 2 kDa to about 150 kDa, and from about 3kDa to about 100 kDa. Table 1 below provides general guidance forselecting the membrane for retention of RNA based on the nucleotidecontent of a nucleic acid. Alternatively, the soluble RNA is retained onthe filter and later recovered. Suitable membranes for soluble RNAretention include anionic membranes such as PVDF.

TABLE 1 NMWL guidelines for selecting semipermeable membrane forultrafiltration. Single-stranded nucleotide Double-stranded nucleotideNMWL cut-off (bases) cut-off (base pair)  3 kDa 10 10 10 kDa 30 20 30kDa 60 50 50 kDa 125 100 100 kDa  300 125

Various commercially available ultrafiltration kits and devices areavailable to concentrating a sample such as Amicon Ultra-4 CentrifugalFilter Units, Amicon Ultra-15 Centrifugal Filter Units, Centricon®centrifugal filter devices (Millipore, Mass., USA), Pierce Concentrator(Thermo Fisher Scientific, IL, USA). In one example, 15 ml of solubleurine fraction can be concentrated to 500 μl using Amicon Ultra-15Centrifugal Filter Units by centrifugation for 30 minutes at 4000× g.

In one example, soluble urine fraction can be concentrated bylyophilization. Lyophilization is a freeze-drying process that works byfreezing the material and then reducing the surrounding pressure andadding enough heat to allow the frozen water in the material to sublimedirectly from the solid phase to gas. Lyophilization machines areavailable from commercial vendors such as Labconco (MO., USA), MillrockTechnology (NY, USA).

In another example, soluble urine fraction can be concentrated byplacing the soluble urine fraction by dialysis against a solutioncontaining polyethylene glycol and using a dialysis bag with appropriatemolecular cutoff. The molecular weight cutoff can range from 3 kDa-100kDa depending on the size of RNA to be retained within the dialysis bag.Appropriate dialysis tubings can be obtained commercially such asSigma-Aldrich, Thermo-Scientific.

Methods for RNA Isolation and Extraction

RNA may be isolated and extracted from aqueous samples such as solubleurine fraction or soluble urine concentrate using standard techniques,see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989), Cold Spring Harbor Press, Plainview, N.Y.Particularly useful are solid phase extraction methods. Reagents andkits for isolating RNA from a biological sample are commerciallyavailable e.g., RNeasy Maxi Kit, RNeasy Protect Mini kit, RNeasy ProtectCell Mini kit, QIAamp RNA Blood Mini kit, from Qiagen; MELT™,RNaqueous®, ToTALLY RNA™, RiboPure™-Blood, Poly(A)Purist™ from AppliedBiosystems; TRIZOL® reagent, Dynabeads® mRNA direct kit from Invitrogen.In one example, kits provided by Qiagen employ silica resin to bindnucleic acid including RNA. RNA in the solution binds to the silicaresin while the proteins and other solutes passes through. After severalsteps of washing, RNA can be eluted using the buffer provided by themanufacturer. In one example, NucliSENS® easyMAG® automated system(bioMérieux, Inc., NC, USA) may be used for the extraction of totalnucleic acids including RNA. RNA from soluble urine fraction or solubleurine concentrate will bind to NucliSENS® magnetic silica particles. TheRNA bound to the magnetic silica particles will be washed with washbuffer supplied by the manufacturer and will be eluted from the magneticsilica particles by heating using manufacturer's protocol. In anotherexample, RNA can be isolated by adsorbing on an anion exchange resinfollowed by elution with high salt buffer. Exemplary anion exchangeresins include Diethylaminoethyl (DEAE) crosslinked to polystyrene orcellulose, DNAPac® series of polymer-based anion-exchange columns fromDionex, anion exchange columns from Thermo Scientific.

Reverse Transcription of RNA to cDNA

Various methods to reverse transcribe RNA to cDNA are known in the art.Various reverse transcriptases may be used, including, but not limitedto, MMLV RT, RNase H mutants of MMLV RT such as Superscript andSuperscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMVRT, and thermostable reverse transcriptase from Thermus Thermophilus. Inone example, RNA extracted from soluble urine fraction or soluble urineconcentrate may be reverse transcribed to cDNA using the protocoladapted from the Superscript II Preamplification system (LifeTechnologies, GIBCO BRL, Gaithersburg, Md., catalog no: 18089-011), asdescribed by Rashtchian, A., PCR Methods Applic. (1994), 4: S83-S91. Themethod is described below.

One (1) to five (5) micrograms of RNA extracted from soluble urinefraction or soluble urine concentrate in 13 μl of DEPC-treated water isadded to a clean microcentrifuge tube. One microliter of either oligo(dT) (0.5 mg/ml) or random hexamer solution (50 ng/μl) is added andmixed gently. The mixture is then heated to 70 degrees centigrade for 10minutes and then incubated on ice for one minute. Then, it iscentrifuged briefly followed by the addition of 2 μl of 10× Synthesisbuffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl, 25 mm magnesium chloride, 1mg/ml of BSA), 1 μl of 10 mM each of dNTP mix, 2 μl of 0.1 M DTT, 1 μlof SuperScript II RT (200 U/μl) (Life Technologies, GIBCO BRL,Gaithersburg, Md.). After gentle mixing, the reaction is collected bybrief centrifugation, and incubated at room temperature for 10 minutes.The tube is then transferred to a 42° C. water bath or heat block andincubated for 50 minutes. The reaction is then terminated by incubatingthe tube at 70° C. for 15 minutes, and then placing it on ice. Thereaction is collected by brief centrifugation, and 1 μl of RNase H (2units) is added followed by incubation at 37° C. for 20 minutes beforeproceeding to nucleic acid amplification.

In another example, reverse transcription of RNA to cDNA was combinedwith the RT-PCR reaction using RNA UltraSense® one-step real-time (RT)PCR System (Invitrogen).

Detection of RNA

The presence or amount of RNA isolated from soluble urine fraction orsoluble urine concentrate can be determined by several methods known inthe art. In one example, RNA can be detected by Northern blot. See,e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition (1989), Cold Spring Harbor Press, Plainview, N.Y. In anotherexample, RNA can be detected by reverse transcription coupled with PCR,including real-time PCR. The cDNA is amplified in a real-time PCRreaction using gene specific primers. Real-time PCR detects the copynumber of PCR templates such as cDNA in a PCR reaction. Exemplarymethods for quantification of RNA by real-time PCR is described by Nolanet al. (Nat Protoc. 2006; 1(3): 1559-82) and Gertsch et al. (Pharm Res.2002 August; 19(8): 1236-43). The references are incorporated herein byreference. In another example, RT-PCR is performed in a combination witha reverse transcription of RNA to cDNA reaction using RNA UltraSense®one-step real-time (RT) PCR System (Invitrogen).

In one example, amplification of cDNA is monitored by SYBR green dye.The dye binds to double-stranded (ds)DNA in PCR, causing fluorescence ofthe dye. An increase in DNA product during PCR therefore leads to anincrease in fluorescence intensity and is measured at each cycle, thusallowing DNA concentrations to be quantified.

In another example, amplification of cDNA is monitored by TaqMan® probes(Heid et al., Genome Res. 1996; 6: 986-994). TaqMan® probes are based onthe principle of fluorescence quenching and involve a donor fluorophoreand a quenching moiety. The term “fluorophore” as used herein refers toa molecule that absorbs light at a particular wavelength (excitationfrequency) and subsequently emits light of a longer wavelength (emissionfrequency). The term “donor fluorophore” as used herein means afluorophore that, when in close proximity to a quencher moiety, donatesor transfers emission energy to the quencher. As a result of donatingenergy to the quencher moiety, the donor fluorophore will itself emitless light at a particular emission frequency than it would have in theabsence of a closely positioned quencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.Suitable quenchers are selected based on the fluorescence spectrum ofthe particular fluorophore. Useful quenchers include, for example, theBlack Hole™ quenchers BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies,Inc.), TAMRA, 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL), andthe ATTO-series of quenchers (ATTO 540Q, ATTO 580Q, and ATTO 612Q;Atto-Tcc GmbH). TaqMan® probes are designed to anneal to an internalregion of a PCR product. When the polymerase (e.g., reversetranscriptase) replicates a template on which a TaqMan® probe is bound,its 5′ exonuclease activity cleaves the probe. This ends the activity ofthe quencher (no FRET) and the donor fluorophore starts to emitfluorescence which increases in each cycle proportional to the rate ofprobe cleavage. Accumulation of PCR product is detected by monitoringthe increase in fluorescence of the reporter dye. If the quencher is anacceptor fluorophore, then accumulation of PCR product can be detectedby monitoring the decrease in fluorescence of the acceptor fluorophore.

To ensure accuracy in the quantification, it is usually necessary tonormalize expression of a target gene to one or more reference genesthat are stably expressed. Exemplary reference genes include beta actin(ACTB), beta-2 microglobulin (B2M), glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Relative concentrations of DNA present during theexponential phase of the reaction are determined by plottingfluorescence against cycle number on a logarithmic scale (so anexponentially increasing quantity will give a straight line). Athreshold for detection of fluorescence above background is determined.The cycle at which the fluorescence from a sample crosses the thresholdis called the cycle threshold, Ct. A lower Ct value indicates highercopy number of an RNA. Amounts of RNA is determined by comparing theresults to a standard curve produced by real-time PCR of serialdilutions of a known amount of RNA or DNA.

Detection by Hybridization. RNA isolated from soluble urine fraction orsoluble urine concentrate can be detected following reversetranscription and amplification by hybridization with a nucleic probethat hybridizes specifically to the RNA of interest (i.e., a targetRNA). The methods of the present invention can incorporate all knownmethods and means and variations thereof for carrying out DNAhybridization, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.

The RNA or cDNA may form a complex on a solid support prior to beingdetected. The complex may comprise a capture probe anchored to a solidsupport, the RNA of interest hybridized to the capture probe, and adetectably labeled probe hybridized to the RNA of interest. In somecases, the solid support may comprise a first member of a binding pairand the capture probe may comprise a second member of the binding pair.The binding of the first member of the binding pair to the second memberof the binding pair may anchor the capture probe to the solid support.Examples of solid support include but are not limited to beads,microparticles, microarray plates, microwells. Examples of binding pairinclude but are not limited to biotin/streptavidin, ligand-receptor,hormone-receptor, and antigen-antibody.

RNA and/or cDNA can be detected by performing an array-basedhybridization to detect the genes of interest in a sample, or todiagnose a disease in an individual. The resolution of array-basedmethod is primarily dependent upon the number, size and map positions ofthe nucleic acid elements within the array, which are capable ofhybridizing to the RNA. Microarrays are available commercially thatcover all human genes. For example, GeneChip® Human Exon 1.0 ST Arrayfrom Affymetrix (CA, USA), Whole Human Genome Microarray Kit fromAgilent Technologies (CA, USA) are capable of evaluating gene expressionof all known transcripts in human.

Alternatively, the hybridized complexes can also be detected using flowcytometry. Flow cytometry is a technique well-known in the art. Flowcytometers hydrodynamically focus a liquid suspension of particles(e.g., cells or synthetic microparticles or beads) into an essentiallysingle-file stream of particles such that each particle can be analyzedindividually. Flow cytometers are capable of measuring forward and sidelight scattering which correlates with the size of the particle. Thus,particles of differing sizes may be used in invention methodssimultaneously to detect distinct nucleic acid segments. In additionfluorescence at one or more wavelengths can be measured simultaneously.Consequently, particles can be sorted by size and the fluorescence ofone or more fluorescent labels probes can be analyzed for each particle.Exemplary flow cytometers include the Becton-Dickenson ImmunocytometrySystems FACSCAN. Equivalent flow cytometers can also be used in theinvention methods.

RNA Associated With Disease

RNA isolated from soluble urine fraction or soluble urine concentratemay be associated with a disease and can be useful for diagnosis andprognosis of such disease. The RNA associated with a disease may beoverexpressed or underexpressed in a disease condition. Detecting thelevel of RNA may be indicative of the diagnosis and prognosis of thedisease. Exemplary RNA associated with a disease that can be isolatedfrom soluble urine fraction or soluble urine concentrate by the methodsof the present invention are shown in Table 2 below.

TABLE 2 RNA associated with disease GenBank Disease Gene Name SymbolAccession No. Reference Prostate Prostate cancer antigen 3 PCA3NR_015342 Mearini et al. Cancer Biomarkers. 2009; 14(4): 235-43 Schalkenet al., Urology. 2003; 62(5 Suppl 1): 34- 43 Tinzl et al. Eur. Urol.2004; 46(2): 182-6 Prostate P antigen family, PAGE4 NM_007003 Iavaroneet al. Mol Cancer member 4 Cancer Ther. 2002; 1(5): 329-35 Kong et al.Hepatogastroenterology. 2004; 51(59): 1519-23 Prostate Solute carrierfamily 45, SLC45A3 NM_033102 Sheridan et al. Am J Cancer member 3 SurgPathol. 2007; 31(9): 1351-5 Prostate Kallikrein-related KLK3NM_001030047 Mabjeesh et al. Prostate. Cancer peptidase 3 2009; 69(11):1235-44 Ovarian Phosphoinositide-3-kinase, PIK3CA NM_006218 Kadota etal. Cancer Cancer catalytic, alpha Res. 2009; 69(18): 7357- polypeptide65) Ovarian Erythroblastic leukemia ERBB2 NM_001005862 Tong et al. BJOG.2009; Cancer viral oncogene homolog 2 116(8): 1046-52 BladderHyaluronoglucosaminidase HYAL1 NM_007312 Eissa et al. Cancer. Cancer 12005; 103(7): 1356-62 Bladder Bladder cancer associated BLCAPNM_001167820 Yao et al. Mol Cell Cancer protein Biochem. 2007; 297(1-2): 81-92 Bladder Solute carrier family 35, SLC35E3 NM_018656 Clark etal. Genome Cancer member E3 Res. 2003; 13(10): 2265- 70 Bladder Tumorprotein p53 TP53 NM_000546 Ouerhani et al. Cancer Cancer Invest. 2009;27(10): 998-1007 Uterine P antigen family, member PAGE4 NM_007003Iavarone et al. Mol Cancer 4 Cancer Ther. 2002; 1(5): 329-35 Kong et al.Hepatogastroenterology. 2004; 51(59): 1519-23 Cervical Suppressor ofST20 NM_001100879 Kim et al. Int J Cancer. Cancer tumorigenicity 20 2002Feb. 20; 97(6): 780- 6

The references and the sequences of the RNA associated with a diseaseare incorporated herein by reference.

Example 1 Detection of RNA from Large Volume of Liquid Sample

The ability to detect RNA in a large volume of liquid sample was testedby adding 5 μl of RNA (845.6 ng/μl) to 15 ml of Tris-EDTA (TE) buffer.The resulting RNA solution was concentrated to less than 500 μl bycentrifugation using Amicon Ultra-15 filter units with nominal molecularweight limit of 3 kDa and 10 kDa (Millipore, Mass., USA). Theconcentration of RNA in the retentate from two different membranefilters were determined using NanoDrop™ spectrophotometer (ThermoScientific), which requires small volume of sample for analysis. Therecovery of RNA were comparable from the two membrane filter types: 94%for 10 kDa and 100% for 3 kDa. The results are shown in Table 3 below.

TABLE 3 Retention of spiked cell line RNA in TE measured by Nanodropconcentration. Volume Conc. Total Sample (μl) (ng/μl) (ng) % RecoverySpiked RNA/15 ml TE 5 845.6 4228 10 kDa membrane retentate 160 24.9 3984 94% 3 kDa membrane retentate 270 15.7 4239 100%

Example 2 Comparison of Recovery of Spiked RNA from Membranes withDifferent Pore Sizes

The range of filter pore sizes that can be used to concentrate the RNAwere evaluated for RNA retention using filter columns ranging from 3kDa-100 kDa. A known amount of cell line RNA (34 g) was spiked into alarge volume of TE buffer (75 ml), split into five aliquots for astarting amount of 6.8 μg of total RNA per 15 ml aliquot. Each 15 mlaliquot was concentrated through five separate filter columns withdifferent pore sizes (nominal molecular weight limit: 3 kDa, 10 kDa, 30kDa, 50 kDa and 100 kDa retention, respectively). After concentrationwith the filter columns, the % recovery was determined. First, RNA yieldwas calculated by multiplying the final volume of sample by the finalconcentration of the sample measured by nanodrop. Second, the RNA yieldwas divided by the starting amount of RNA (6.8 μg) to give the final %recovery of each filter column. Based on these results, the 3 kDa poresize gave the highest recovery of 94%, followed by 10 kDa (87%), 30 kDa(78%), 50 kDa (80%), and 100 kDa with the lowest and final yield (67%).The results are shown in Table 4 below.

TABLE 4 Retention of spiked cell line RNA in TE measured by Nanodropconcentration. Volume Conc. Total Sample (μl) (ng/μl) (ng) % RecoverySpiked RNA/15 ml TE 5 1360 6800 100 kDa membrane retentate 135 33.7 455067% 50 kDa membrane retentate 206 26.4 5438 80% 30 kDa membraneretentate 290 18.4 5336 78% 10 kDa membrane retentate 190 31.3 5947 87%3 kDa membrane retentate 428 15.0 6420 94%

Example 3 Comparison of Recovery of Endogenous Urine RNA from Membraneswith Different Pore Sizes

Some factors may effect the efficiencies in retention of RNA in a realsample with endogenous RNA versus a sample spiked with RNA. Thesefactors include the presence of partially degraded or fragmented RNA andthe presence of urine RNases that may degrade RNA prior to processing.The ability of membranes with different pore sizes to retain endogenousurine RNA was evaluated. Whole urine (75 ml) was obtained from fiveseparate donors and split into five 15 ml aliquots per donor. Each ofthe five aliquots per donor was concentrated using the filters of fivedifferent pore sizes (nominal molecular weight limit: 3 kDa, 10 kDa, 30kDa, 50 kDa, and 100 kDa). RNA from each sample of concentrated urinewas extracted using EasyMag. Amplification of two different transcripts(GAPDH and ABL1) was performed for each sample by RT-PCR. In order toquantitate retention efficiencies, RNA concentrated from the topperforming filter column in the RNA spiking studies (3 kDa) was used asthe baseline (100%) for each donor. Using the cycle threshold (Ct)obtained by qRT-PCR, the recoveries for the 10 kDa-100 kDa filtercolumns were calculated based on the 3 kDa Ct values that were set at100%. Based on these results, retention of endogenous RNA was dependenton both the donor and the transcript, with all pore sizes above 3 kDademonstrating significantly reduced efficiency. The average retentionfor pore sizes 10 kDa-100 kDa for transcript 1 ranged from 32%-47% and16%-31% for transcript 2. The results are shown in Table 5 below.

TABLE 5 Recovery of endogenous urine RNA using filter column membraneswith different pore sizes. Transcript 1 Transcript 2 Donor 3 kDa 10 kDa30 kDa 50 kDa 100 kDa 3 kDa 10 kDa 30 kDa 50 kDa 100 kDa 1 100% 66% 59%79% 43% 100% 25% 39% 36% 9% 2 100% 67% 69% 79% 59% 100% 47% 40% 50% 19%3 100% 40% 43% 24% 25% 100% 28% 28% 9% 12% 4 100% 26% 20% 10% 4% 100%26% 25% 12% 8% 5 100% 38% 24% 34% 28% 100% 27% 11% 13% 31% Avge 100% 47%43% 45% 32% 100% 31% 29% 24% 16%

Example 4 Sample Preparation and RNA Extraction from Urine Samples

Urine sample (30 ml) was obtained from an individual with benignprostate hyperplasia was split into two 15 ml aliquots for extraction ofRNA from cellular components of urine sediment and soluble urinefractions.

The first aliquot of urine was processed for RNA extraction from thecells in the urine sediment using ZR Urine RNA Isolation Kit™ (ZYMOResearch Corporation). Briefly, cells were separated from urine by asyringe filter. The cells were retained on the syringe filter and thefiltrate was collected separately. The retained cells were lyseddirectly on the filter using 700 μl of RNA Extraction Buffer Plus™reagent (ZYMO Research Corporation) and the cell lysate was collected ina 1.5 ml tube. The cell lysate was mixed with an equal volume of ethanoland passed through Zima-Spin IC™ column. The column was washed with 300μl of RNA Wash Buffer. Total RNA was eluted from the column by applying25 μl of the supplied RNA Elution Buffer directly to the column membranefollowed by centrifugation.

The filtrate collected from the syringe filtration step described abovewas further concentrated using Amicon Ultra-15, nominal molecular weightlimit of 3 kDa (Millipore, Mass., USA) to a final filtrate volume of 500μl (soluble urine concentrate), representing approximately a 30-foldconcentration. The total nucleic acid was extracted from the solubleurine concentrate using NucliSENS® easyMAG® (bioMérieux, Inc., NC, USA)using manufacturer's protocol. Briefly, total nucleic acid binds toNucliSENS® magnetic silica particles. The magnetic silica particles wereseparated from the liquid portion using a magnetic field. The nucleicacid bound to silica particles were washed with the wash buffer providedby the manufacturer. The nucleic acid is finally released from the solidphase with the elution buffer. FIG. 1 (pathways 1 and 2) shows aschematic of the experimental design used to process the first urinealiquot (ultrafiltration step not shown).

The second urine aliquot (15 ml) was directly applied to an AmiconUltra-15, having a nominal molecular weight limit of 3 kDa (Millipore,Mass., USA). The urine sample was concentrated to 500 μl. The totalnucleic was extracted from the soluble urine concentrate usingNucliSENS® easyMAG® (bioMérieux, Inc., NC, USA) using manufacturer'sprotocol as discussed above. FIG. 1 (pathway 3) shows a schematic of theexperimental design used to process the second urine aliquot(ultrafiltration step not shown).

Example 5 cDNA Synthesis and RT-PCR

cDNA Synthesis and RT-PCR were performed in a one-step process using RNAUltraSense® one-step real-time (RT) PCR System (Invitrogen): First, RNAwas treated with DNase to eliminate DNA using RNA-free (Ambion). Amaster mix was prepared with following components for each reaction: RNAUltraSense Enzyme Mix 2.5 RNA UltraSense 5× Reaction Mix 10 μl, Taqmanprobe primer pair (10 μM concentration each) 1 μl, Fluorogenic probe (10μM) 1 μl, ROX Reference Dye 1 μl. Next, 3 μl of RNA template in 31.5 μlof DEPC-treated water per reaction was added to a clean microcentrifugetube. The 34.5 μl of template was added to the 15.5 μl of Master mix fora total of 50 μl for each reaction. After gentle mixing, the reactionmixture was subjected to brief centrifugation, and was placed in apreheated programmed thermal cycler. The instrument was programmed toperform cDNA synthesis immediately followed by PCR amplification usingthe following cycling parameters: 50° C. for 15 minute hold, 95° C. for2 minute hold, 40-50 cycles of: 95° C. for 15 seconds and 60° C. for 30seconds. After cycling, the reaction was held at 4° C. until furtheranalysis.

Example 6 Estimation of Gene Expression Levels

Expression levels of four genes: heat shock 60 kDa protein 1 (HSPD1),inosine monophosphate dehydrogenase 2 (IMPDH2), PDZ and LIM domain 5(PDLIM5), and UDP-N-acteylglucosamine pyrophosphorylase 1 (UAP1); andfive reference genes: c-abl oncogene 1, receptor tyrosine kinase (ABL1),beta actin (ACTB), beta-2 microglobulin (B2M),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta glucuronidase(GUSB) were evaluated using the RNA UltraSense® one-step RT-PCR System.Taqman® probes were used to monitor DNA synthesis. Fluorescent signalswere measured and plotted against the number of PCR cycles. The Ctvalue, the point at which the fluorescence crosses the baselinethreshold is measured for each gene. A lower Ct value indicates higherinitial concentration of template DNA and therefore initial RNA. The Ctvalues for four test genes and five reference genes were determinedusing the RNA isolated from cells present in urine, the urinesupernatant which is free of cells, and whole urine without furtherseparation of cells. The Ct values of different genes in various samplesare presented in Table 6 below.

TABLE 6 Ct values of the Genes in Different Samples Test Genes 5Reference Genes Kit Sample HSPD1 IMPDH2 PDLIM5 UAP1 ABL1 ACTB B2M GAPDHGUSB Zymo Urine 29.9 26.7 31.0 33.3 32.6 24.6 25.1 23.1 30.8 SedimentAmicon Urine 26.0 26.8 28.3 31.9 30.7 23.9 26.5 22.1 28.8 SupernatantSoluble 26.6 24.8 27.1 30.9 29.8 21.4 23.0 20.6 27.3 Urine Concentratefrom Whole Urine (3K) Soluble 29.2 28.2 30.5 32.8 33.2 24.6 27.0 24.430.3 Urine Concentrate from Whole Urine (10K)

The results in Table 6 demonstrate that the amount of RNA in whole urineis generally higher than that obtained from the cells present in urine.Additionally, the urine supernatant contains more transcript than thecells in urine sediment for the majority of the genes tested.Furthermore, the expression pattern of the five reference genes variedamong the cells in urine sediment, urine supernatant (after separationof cells) and soluble urine concentrate (without separation of cells).As seen in Table 6, concentration of whole urine to form a soluble urineconcentrate consistently yielded higher amounts of RNA when a membranewith smaller pore size was used (cf. MW=3 K versus MW=10 K cutoff).

A score matrix for the four test genes were created and normalized bythe five reference genes. The normalized four test genes scores indicatethe expression pattern of the genes are different in the differentfractions of urine (Table 7).

TABLE 7 Normalized Gene Scores 4-gene score normalized by reference geneindicated RNA source AVG ABL ACTB B2M GAPDH GUSB Urine Sediment −8.3 6.6−15.6 −14.2 −19.8 1.6 (cellular RNA) Urine Supernatant −5.1 6.8 −12.1−4.9 −17.1 1.5 (after separation of cells) Soluble Urine −8.1 6.8 −16.5−12.1 −18.7 −0.1  Concentrate (without separation of cells)

Example 7 Rate of Sufficient RNA Quantity for Gene Expression AssayBased on Urine Fraction and Time-to-Process

Urine samples were collected from 61 donors that were received atvarying time points post collection (<28 hrs n=32, 48 hrs n=17, >65 hrsn=12). Effects of time-to-process (i.e. time from collection to fractionseparation and extraction) on RNA quantity and quality were determinedby calculating the success rates of samples in each time point range.Success rates were determined by the amount of GAPDH transcript presentin the RNA sample as measured by real-time RT-PCR. At <28 hourstime-to-process, most samples (84-97%) yielded sufficient quantities foranalysis of gene expression for all three sample types. When longertime-to-process occurred (>48 hours), urine sediment RNA is largelyunsuccessful (41-50% success rate) as compare to urine supernatant andwhole urine. Whole urine had a higher success rate at 48 hours (94%)than urine supernatant (76%), but urine supernatant maintained a closesuccess rate even beyond 65 hours (67%) than whole urine (50%). See FIG.2.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.

Thus, it should be understood that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

What is claimed is:
 1. A method for processing urine for detection ofRNA expression associated with a cancer, comprising: a) separating urinesediment from the soluble urine fraction of a urine sample obtained froman individual suspected of having cancer, wherein RNA associated withthe cancer is present in the soluble urine fraction; and b)concentrating the soluble urine fraction by ultrafiltration to produce asoluble urine concentrate, wherein the volume of the soluble urineconcentrate is reduced at least 50% from the original urine volume,wherein the cancer-associated RNA is selected from the group consistingof prostate cancer antigen 3 (PCA3), p antigen family, member 4 (PAGE4),solute carrier family 45, member 3 (SLC45A3), kallikrein-relatedpeptidase, member 3 (KLK3), phosphoinositide-3-kinase, alpha polypeptide(PIK3CA), erythroblastic leukemia viral oncogene homolog 2 (ERBB2),hyaluronoglucosaminidase (HYAL1), bladder cancer associated protein(BLCAP), solute carrier family 35, member E3 (SLC35E3), tumor proteinp53 (TP53), and suppressor of tumorigenicity 20 (ST20).
 2. The method ofclaim 1, wherein the cancer is prostate cancer, ovarian cancer, bladdercancer, uterine cancer or cervical cancer.
 3. The method of claim 1,wherein urine sediment is separated from the soluble urine fraction bycentrifugation or filtration.
 4. The method of claim 1, wherein saidultrafiltration comprises a filter having a nominal molecular weightlimit of not more than about 100,000 daltons.
 5. The method of claim 1,wherein said ultrafiltration comprises a filter having a nominalmolecular weight limit of not more than about 50,000 daltons.
 6. Themethod of claim 1, wherein said ultrafiltration comprises a filterhaving a nominal molecular weight limit of between about 3,000 daltonsand about 10,000 daltons.
 7. The method of claim 1, wherein saidultrafiltration comprises a filter having a nominal molecular weightlimit of about 3,000 daltons.
 8. The method of claim 1, wherein the RNAis isolated from the soluble urine concentrate by solid phaseextraction.
 9. The method of claim 1, further comprising: c) isolatingRNA from the soluble urine concentrate produced in step (b), d) reversetranscribing the RNA from the soluble urine concentrate produced in step(c) to form cDNA; and e) amplifying the cDNA produced in step (d). 10.The method of claim 9, wherein urine sediment is separated from thesoluble urine fraction by centrifugation or filtration.
 11. The methodof claim 9, wherein said ultrafiltration comprises a filter having anominal molecular weight limit of not more than about 50,000 daltons.12. The method of claim 9, wherein said ultrafiltration comprises afilter having a nominal molecular weight limit of between about 3,000daltons and about 10,000 daltons.
 13. The method of claim 9, whereinsaid ultrafiltration comprises a filter having a nominal molecularweight limit of about 3,000 daltons.
 14. The method of claim 9, whereinthe RNA is isolated from the soluble urine concentrate by solid phaseextraction.
 15. The method of claim 9, wherein the RNA is mammalian RNA.16. A method for isolating nucleic acids from a urine sample fordetection of RNA expression associated with a cancer, said methodcomprising, a) providing a urine sample from an individual suspected ofhaving a cancer, b) processing said urine sample to produce a cell-freeurine sample, wherein RNA associated with the cancer is present in thecell-free urine sample, and c) subjecting said cell-free urine sample toultrafiltration using a filter having a nominal molecular weight limitof between about 3 kDa and about 100 kDa, to produce a soluble urineconcentrate, wherein the RNA associated with the cancer is retained inthe soluble urine concentrate, and wherein the volume of the solubleurine concentrate is reduced at least 50% from the original urinevolume, wherein the benign prostate hyperplasia-associated RNA isselected from the group consisting of prostate cancer antigen 3 (PCA3),p antigen family, member 4 (PAGE4), solute carrier family 45, member 3(SLC45A3), kallikrein-related peptidase, member 3 (KLK3),phosphoinositide-3-kinase, alpha polypeptide (PIK3CA), erythroblasticleukemia viral oncogene homolog 2 (ERBB2), hyaluronoglucosaminidase(HYAL1), bladder cancer associated protein (BLCAP), solute carrierfamily 35, member E3 (SLC35E3), tumor protein p53 (TP53), and suppressorof tumorigenicity 20 (ST20).
 17. The method of claim 16, wherein thecancer is prostate cancer, ovarian cancer, bladder cancer, uterinecancer or cervical cancer.
 18. The method of claim 16, furthercomprising: d) isolating RNA from the soluble urine concentrate producedin step (c), e) reverse transcribing the RNA from the soluble urineconcentrate produced in step (d) to form cDNA; and f) amplifying thecDNA produced in step (e).
 19. The method of claim 18, wherein the RNAis isolated from the soluble urine concentrate by solid phaseextraction.
 20. A method for processing urine for detection of RNAexpression associated with a cancer comprising concentrating a urinesample from an individual suspected of having a cancer to produce asoluble urine concentrate wherein RNA associated with the cancer presentin the urine sample is retained in the soluble urine concentrate,wherein the volume of the soluble urine concentrate is reduced at least50% from the original urine volume, and wherein the cancer-associatedRNA is selected from the group consisting of prostate cancer antigen 3(PCA3), p antigen family, member 4 (PAGE4), solute carrier family 45,member 3 (SLC45A3), kallikrein-related peptidase, member 3 (KLK3),phosphoinositide-3-kinase, alpha polypeptide (PIK3CA), erythroblasticleukemia viral oncogene homolog 2 (ERBB2), hyaluronoglucosaminidase(HYAL1), bladder cancer associated protein (BLCAP), solute carrierfamily 35, member E3 (SLC35E3), tumor protein p53 (TP53), and suppressorof tumorigenicity 20 (ST20).